The DNA of a cell presents the first line of control over metabolic and genetic processes. Replication of this repository of information is the primal biochemical act of cells. The goals of this proposal are to determine how this event occurs. Our focus will be on the function of DNA polymerases in E. coli. We will use biochemical and genetic means to analyze a mutation (pcbA) which permits by-pass of a replication block due to a defective DNA polymerase III through utilization of DNA polymerase I. We will investigate the biochemical mechanism of action of the pcbA mutation using in vitro complementation in a phage replication system. Characterization of the pcb gene will also require cloning, using complementation of temperature sensitivity in polCts mutants. Achieving this goal will permit biochemical analysis of the mutant gene and study of the gene product. The relief of replication blockade suggests that DNA polymerase III may be eliminated by mutagenesis. We will attempt to do that. We will also purify and characterize the DNA polymerase I arising as a reaction of suppression of the polAI amber mutation.